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Cytogenetics
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Cytogenetics is the study of chromosomes and their role in heredity.
The goal of cytogenetics:
1. diagnosis of chromosomal abnormalities.
2. localisation of any (often abnormal) chromosomal region/DNA sequence.
Cytogenetics
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Chromatin: non condensed DNA with proteins attached (interphase of the cell cycle)
Chromosome: condensed DNA with proteins attached (M phase of the cell cycle)
Human cells contain 46 chromosome, 44 autosome and 2 sex chromosome.
A karyotype is the number and appearance of chromosomes in the nucleus of a eukaryotic cell. The term is also used for the complete set of chromosomes in a species, or an individual organism.
The chromosomes are depicted (by rearranging a microphotograph) in a standard format known as a karyogram or idiogram: in pairs, ordered by size and position of centromere for chromosomes of the same size.
Basic Cytogenetic Terms
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Human chromosomes are examined in dividing cells (bone marrow/placental cells, lymphocytes).
The cell cycle and the detection of the chromosomes
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1. chr. 1. chr. (human)(human)
DNA length 50 mm
chr. length 3-4 µm
10.000 x 10.000 x condensationcondensation
The steps of chromosome assembly
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The steps of chromosome assembly
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The history of human chromosome identification
1879. Arnold: First visualization of human chromosomes.
1888. Waldeyer: The word chromosome (chroma: color, soma: body)
1882. Walther Flemming: 20-28 chromosomes in cells of cornea
1921. T.S. Painter: 48 human chromosomes, X & Y chromosomes (Science)
1956. Jo Hin Tijo és Albert Levan : 46 human chromosomes (Hereditas)
1959. Lejeune: trisomy 21=Down syndrome
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1960. Denver: chromosomes s numbered ((1-22) based on their size
1963. London: chromosome grouping (A-G)
1966. Chicago: big chromosome syndromes
1971. Paris, 1976. Mexico, 1978. Stockholm: chromosome banding
1995. ISCN : International System for Human Cytogenetic Nomenclature
Kariotyping conferences
The history of human chromosome identification
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46, XX
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46, XY
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Chromosome structure
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Chromosome bandingThe chromosome banding techniques use different chemicals The chromosome banding techniques use different chemicals
to stain the chromosomes in order to identify them or to show to stain the chromosomes in order to identify them or to show
chromosomal rearrangements. Based on the stains used the chromosomal rearrangements. Based on the stains used the
following banding techniques exist:following banding techniques exist:
•• GG--banding:banding: Giemsa staining
•• RR--banding:banding: (reverse) modified Giemsa staining
•• CC--banding:banding: centromer specific staining
•• TT--banding:banding: telomer specific staining
•• QQ--bandingbanding: : quinacrin staining (fluorescent)Starting from the centromere, the short and the long chromosome arms are numbered based on the banding pattern.
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QQ--bandingbandingG-banding
R-banding C-banding T-banding
Chromosome banding
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Human diseases resulting from altered kariotype are mostly caused by ANEUPLOIDIES, TRANSLOCATIONS, and DELETIONS.
Chromosomal abnormalities
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ANEUPLOIDY
ANEUPLOIDY is the abnormal number of chromosomes. The most frequent aneuploidia is trisomy, having three instead of two chromosomes.
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MITOTIC NON-DISJUNCTION
MEIOTICNON-DISJUNCTION
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MEIOTIC NON-DISJUNCTION—> ANEUPLOIDY
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MEIOTIC/MITOTIC NON-DISJUNCTION
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Chromosome number abnormalitiesEuploid chromosome mutation: Triploidity,Triploidity, PoliploidPoliploidityityAneuploid chromosome mutationAutosomal occurence Sex chromosomal occurence
Trisomy21. trisomyDown-syndrome
1/700 47, XXX Triplo-X-syndrome
1/1000 female
18. trisomyEdwards- syndrome
1/13000 47, XXY Klinefelter-syndrome
1/1000 male
13. trisomyPatau-syndrome
1/15000 47, XYY Dupla-Y-syndrome
1/1000 male
Rare trisomies 3-,7-,8-,9-,12-,14-,15-,19-,22-esMonosomy 45, X0 Turner-
syndrome1/2500 female
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AUTOSOMAL ANEUPLOIDIES: DOWN SYNDROME
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AUTOSOMAL ANEUPLOIDIES: DOWN SYNDROME
trisomy of the 21. chromosome
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MATERNAL AGE & THE DOWN SYNDROME
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trisomy of the 18. chromosome
AUTOSOMAL ANEUPLOIDIES: EDWARDS SYNDROME
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SEX CHROMOSOMAL ANEUPLOIDIES:
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25Karyotype: 45X0
SEX CHROMOSOMAL ANEUPLOIDIES:TURNER SYNDROME
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SEX CHROMOSOMAL ANEUPLOIDIES:KLINEFELTER SYNDROME
Karyotype: 47XXY
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SEX CHROMOSOMAL ANEUPLOIDIES:TRIPLE X SYNDROME
Karyotype: 47XXX
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SEX CHROMOSOMAL ANEUPLOIDIES:YY SYNDROME
Karyotype: 47XYY
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CLASSIFICATION OF STRUCTURAL CHROMOSOME ABNORMALITIES BASED ON THE NUMBER OF
CHROMOSOMAL BREAK POINTS
terminális deléció
1 törés
reciprok transzlokáció centrikus fúzióvagy
Robertson-féletranszlokáció
2 különböző kromoszómán
paracentrikus inverzió
uazon a kromoszómakaron
gyűrű kromoszóma pericentrikus inverzió
ellentétes kromoszómakaron
uazon a kromoszómán
2 törés
inszerció
3 törés2 különböző
kromoszómán
szerkezeti kromoszóma aberráció
3 breaks3 breaks2 breaks2 breaks1 break1 breakOn 2 different On 2 different
chromosomeschromosomes
Structural abberation of chromosomesStructural abberation of chromosomes
Terminal deletionTerminal deletion On 2 different chromosomesOn 2 different chromosomes InsertionInsertionOn same chromosomeOn same chromosome
Reciprocal Reciprocal translocationtranslocation
On same chromosome On same chromosome armarm
Central fusionCentral fusion
or or
Robertson Robertson translocationtranslocation
On opposite On opposite chromosome armchromosome arm
Paracentric Paracentric inversioninversion
Ring Ring chromosomechromosome
Pericentric Pericentric inversioninversion
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DELETION
A piece of the chromosome is lost
Deletion mapping
Intersticial deletion - Prader-Willi; Angelman syndromes del 5(q11-13)Williams syndrome
Terminal deletion - telomer is lost too – severe symptomsCri du chat syndrome
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TRANSLOCATION
Reciprocal translocation:Reciprocal translocation: breakpoints are on 2 homologous or on 2 different chromosomes
break points are mostly in non coding regions
balanced translocation
breakpoints inside genesbreakpoints inside genes - genes with altered function - genes with altered activity- genes with altered expression- genes with loss of function
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RING CHROMOSOME
forms after tforms after telomere deletion
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INVERSION
Paracentric inversion Pericentrikus inversion
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INHERITED DELETIONSINHERITED DELETIONS
DELETIONDELETION SYMPTOMSYMPTOM
5p5p Mental retardation, cardiac dev. problems
1818pp Physical and mental retardation
XXqq (Fragil(Fragile e X)X) Deformed face, autism
2222qq Thyroid gland/ parathyroid gland development defect
1313qq Tumors, retinoblastoma
INHERITED TRANSLOCATIONSINHERITED TRANSLOCATIONS
TRANSLOCATIONTRANSLOCATION SYMPTOMSYMPTOM
44--20. chromosome20. chromosome mental retardation, deformed face
X X -- 13. chromosome13. chromosome mental retardation
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ACQUIRED DELETIONS IN TUMORSACQUIRED DELETIONS IN TUMORS
DELETIONDELETION TUMORTUMOR
APCAPC--genegene colorectal
Rb (Retinoblastoma) geneRb (Retinoblastoma) gene any tumor
P53 geneP53 gene any tumor
22. chromosome22. chromosome acute myeloid leukemia
TRANSLOCATIONTRANSLOCATION TUMORTUMOR
99--22 chr. (Philadelphia 22 chr. (Philadelphia chromosome)chromosome)
Chronic myeloid leukemia
88--14 chromosome14 chromosome Burkitt - lymphoma
88--21 chromosome 21 chromosome acute myeloid leukemia
ACQUIRED TRANSLOCATIONS IN TUMORSACQUIRED TRANSLOCATIONS IN TUMORS
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most frequent inherited most frequent inherited mental retardationmental retardation
symptoms:symptoms:-- big head, elongated big head, elongated face, big earsface, big ears-- mild mild –– severe mental severe mental retardationretardation
Xq27.3 Xq27.3 ––increased fragilityincreased fragility
X fra(X) fra(X) YX fra(X) fra(X) Y
FRAGILE X SYNDROME
Xq27.3Xq27.3
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A mutation in the FMRA mutation in the FMR--11 gene gene (Xq28) Xq28)
CGG trinucleotidCGG trinucleotid--repeatrepeat expansionexpansion
5’ UTR affected5’ UTR affected
repeatrepeat expansion is followed by methylation and inhibited expressionexpansion is followed by methylation and inhibited expression
FMRFMR--1 protein is a RNA1 protein is a RNA--binding proteinbinding protein
For the mental retardation the For the mental retardation the mGluR5 mGluR5 ((metabotrmetabotropic opic glutamate receptorglutamate receptor) might be) might beresponsible
FRAGILE X SYNDROME
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CGGCGG
CGGCGG
55--50 repeat50 repeat
CGGCGG
5050--200 repeat200 repeat
200 200 -- repeatrepeat
healthyhealthy
‘Pre‘Pre--mutation’mutation’
‘full mutation’‘full mutation’
FMRFMR--1 gene1 geneFRAGILE X SYNDROME
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FISH: FLUORESCENT IN SITU HIBRIDISATION
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FISHFISH
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Direct and indirect methodDirect and indirect method
Excitation lightExcitation light
FluorescenceFluorescence
Fluorescencently Fluorescencently labeled antibodylabeled antibody
NonNon--fluorescently (DIG) fluorescently (DIG) labeled probelabeled probe
Fluorescently labeled Fluorescently labeled probeprobe
Detectable denaturated DNADetectable denaturated DNA
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FISHFISH
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Types of FISH probesTypes of FISH probeschromosome specific probes: for detection of chromosome number abnormalities
X (red) and Y (green) chromosome specific probes in healthy male and female cells
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whole chromosomes are highlighted with the painting probespainting probes – good for detecting translocations
1. chromosome (left), 22. chromosome (middle), 3. chromosome (right) shown with painting probes
Types of FISH probesTypes of FISH probes
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sex chromosome identification with painting probes. two X chromosome (left) in a female cell, a Y chromosome (right) in a male cell.
Types of FISH probesTypes of FISH probes
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the locus/gene specific probes are used to detect any gene of interest, frequently absence or amplification of tumor suppressors of oncogenes
RB1 gene on the 13. chromosome
Types of FISH probesTypes of FISH probes
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Locus/gene specific FISH probesLocus/gene specific FISH probes•• PraderPrader--Willi syndrome 15q11Willi syndrome 15q11--1313•• Angelman syndrome Angelman syndrome 15q1115q11--1313•• DiDi--George syndrome /VCFS 22q11.2George syndrome /VCFS 22q11.2•• Williams syndromeWilliams syndrome 7q11.237q11.23•• WolfWolf--Hirschhorn syndromeHirschhorn syndrome 4p16.34p16.3•• Cri du Chat syndrome Cri du Chat syndrome Xp22.3Xp22.3•• SRY gene Yp11.3 SRY gene Yp11.3 •• XX--linked ichthyosislinked ichthyosis Xp22.3Xp22.3•• Retinoblastoma (RbRetinoblastoma (Rb--gene) 13q14gene) 13q14•• SmithSmith--Magenis syndromeMagenis syndrome 17p11.217p11.2•• MillerMiller--Dieker syndromeDieker syndrome 17p13.317p13.3
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4821 trisomy: Down-syndrome
Medical application of FISHMedical application of FISH
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gene specific FISH probes proved amplification of oncogenes (erb-B2, EGFR, myc) and deletion of tumor supressor genes (p53, Rb) in a vast range of tumors
The p53 tumorsuppressor gene shown by gene specific FISH probe in leukaemia cells.
Medical application of FISHMedical application of FISH
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The Philadelphia chromosome is frequently occuring in chronic myeloid leukaemia (CML). Diagnosis is based on gene specific FISH probes.
Philadelphia chromosome(Ph1)
abl (Abelson cluster region) gene–encodes a tirosin kinase
bcr (breakpoint cluster region)
Medical application of FISHMedical application of FISH
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A BCR gene is on the 22.,the ABL gene is on the 9. chromosome. Translocation results in fusion of the two genes.
Medical application of FISHMedical application of FISH
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Telomer specific FISH
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X-CHROMOSOME INACTIVATION
Barr Body rule: #BB = #X-1DOSAGE COMPENSATION
XXX:Two Barr Bodies
Heterochromatinization:
Xist RNA
DNA methylation
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X-CHROMOSOME INACTIVATION
RANDOMDURING EARLY EMBRYOGENESISIRREVERSIBLE
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X-CHROMOSOME INACTIVATION
THERE ARE GENES EXPRESSED ON THE INACTIVE X CHROMOSOME!
XX XO
?
THE ROLE OF PSEUDOAUTOSOMAL REGIONS (PRESENT ON BOTH X & Y):CHIASMATA FORMATION, TO ENSURE PROPER SEGREGATION OF X & Y CHROMOSOMES
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XX--CHROMOSOME INACTIVATIONCHROMOSOME INACTIVATION--MOMOSSAIAICISMCISM
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identify the following genotypesidentify the following genotypes
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59Turner syndrome (XO) Turner syndrome (XO)
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60Down syndrome (21 trisomy) Down syndrome (21 trisomy)
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61Klinefelter syndrome (XXY) Klinefelter syndrome (XXY)
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Normal woman (XX) Normal woman (XX)
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63Normal man (XY) Normal man (XY)
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